Antibodies which activate an erythropoietin receptor

ABSTRACT

Antibodies and fragments thereof which activate an erythropoietin receptor and stimulate erythropoiesis are described. Also described are hybridoma cell lines which produce the antibodies and methods and compositions for the treatment of anemia.

FIELD OF THE INVENTION

This invention relates to antibodies which recognize an erythropoietinreceptor. More particularly, the invention relates to antibodies whichactivate an erythropoietin receptor and stimulate erythropoiesis.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein hormone involved in the growthand maturation of erythroid progenitor cells into erythrocytes. EPO isproduced by the liver during fetal life and by the kidney of adults andstimulates the production of red blood cells from erythroid precursors.Decreased production of EPO, which commonly occurs in adults as a resultof renal failure, leads to anemia. EPO has been produced by geneticengineering techniques involving expression and secretion of the proteinfrom a host cell transfected with the gene encoding erythropoietin.Administration of recombinant EPO has been effective in the treatment ofanemia. For example, Eschbach et al. (N. Engl J Med 316, 73 (1987))describe the use of EPO to correct anemia resulting from chronic renalfailure.

The purification of human urinary EPO was described by Miyake et al. (J.Biol. Chem. 252, 5558 (1977)). The identification, cloning, andexpression of genes encoding erythropoietin is described in U.S. Pat.No. 4,703,008 to Lin. A description of a method for purification ofrecombinant EPO from cell medium is included in U.S. Pat. No. 4,667,016to Lai et al.

Little is known about the mechanism by which EPO stimulateserythropoiesis. While it is clear that EPO activates cells to growand/or differentiate by binding to specific cell surface receptors, thespecific mechanism of activation as well as the structure of thereceptor and any associated protein(s) is not completely understood. Theerythropoietin receptor (EPO-R) is thought to exist as a multimericcomplex. Sedimentation studies suggested its molecular weight is 330±48kDa (Mayeux et al. Eur. J. Biochem. 194, 271 (1990)). Crosslinkingstudies indicated that the receptor complex consists of at least twodistinct polypeptides, a 66-72 kDa species, and 85 and 100 kDa species(Mayeux et al. J. Biol. Chem. 266, 23380 (1991)); McCaffery et al. J.Biol. Chem. 264, 10507 (1991)). A distinct 95 kDa protein was alsodetected by immunoprecipitation of EPO receptor (Miura & Ihle Blood 81,1739 (1993)). Another crosslinking study revealed three EPO containingcomplexes of 110, 130 and 145 kDa. The 110 and 145 kDa complexescontained EPO receptor since they could be immunoprecipitated withantibodies raised against the receptor (Miura & Ihle, supra). Expressionof a carboxy-terminal truncated EPO receptor resulted in detection ofthe 110 kDa complex but not the 145 kDa complex. This suggests that thehigher molecular weight complex contains polypeptides present in the 110kDa complex and an additional 35 kDa protein.

Further insight into the structure and function of the EPO receptorcomplex was obtained upon cloning and expression of the mouse and humanEPO receptors (D'Andrea et al. Cell 57, 277 (1989); Jones et al. Blood76, 31 (1990); Winkelmann et al. Blood 76, 24 (1990); PCT ApplicationNo. WO90/08822; U.S. Pat. No. 5,278,065 to D'Andrea et al.) Thefull-length human EPO receptor is a 483 amino acid transmembrane proteinwith an approximately 224 amino acid extracellular domain and a 25 aminoacid signal peptide. The human receptor shows about an 82% amino acidsequence homology with the mouse receptor. The cloned full length EPOreceptor expressed in mammalian cells (66-72 KDa) has been shown to bindEPO with an affinity (100-300 nM) similar to that of the native receptoron erythroid progenitor cells. Thus this form is thought to contain themain EPO binding determinant and is referred to as the EPO receptor. The85 and 100 KDa proteins observed as part of a cross-linked complex aredistinct from the EPO receptor but must be in close proximity to EPObecause EPO can be crosslinked to them. The 85 and 100 KDa proteins arerelated to each other and the 85 KDa protein may be a proteolyticcleavage product of the 100 KDa species (Sawyer J. Biol. Chem. 264,13343 (1989)).

A soluble (truncated) form of the EPO receptor containing only theextracellular domain has been produced and found to bind EPO with anaffinity of about 1 nM, or about 3 to 10-fold lower than the full-lengthreceptor (Harris et al. J. Biol. Chem. 267, 15205 (1992); Yang & JonesBlood 82, 1713 (1993)). The reason for the reduced affinity as comparedto the full length protein is not known. There is a possibility thatother protein species may also be part of the EPOR complex andcontribute to EPO binding thus increasing the affinity. In support ofthis possibility is the observation of Dong & Goldwasser (Exp. Hematol.21, 483 (1993)) that fusion of a cell line with a low affinity EPOreceptor with a CHO cell which does not bind EPO resulted in a hybridcell line exhibiting high EPO binding affinity of the receptor for EPO.In addition, transfection of a full length-EPOR into CHO cells resultedin a cell line with both high and low affinity receptors as measured byScatchard analysis. Amplification of the EPOR copy number increased thelow affinity but not high affinity binding. These results are consistentwith the presence of a limited quantity of a protein present in CHOcells that converts the low affinity EPOR to high affinity.

Activation of the EPO receptor results in several biological effects.Three of the activities include stimulation of proliferation,stimulation of differentiation and inhibition of apoptosis (Liboi et al.Proc. Natl. Acad. Sci. USA 90, 11351 (1993); Koury Science 248, 378(1990)). The signal transduction pathways resulting in stimulation ofproliferation and stimulation of differentiation appear to be separable(Noguchi et al. Mol. Cell. Biol. 8, 2604 (1988); Patel et al. J. Biol.Chem. 267, 21300 (1992); Liboi et al. ibid). Some results suggest thatan accessory protein may be necessary for mediating the differentiationsignal (Chiba et al. Nature 362, 646 (1993); Chiba et al. Proc. Natl.Acad. Sci. USA 90, 11593 (1993)). However there is controversy regardingthe role of accessory proteins in differentiation since a constitutivelyactivated form of the receptor can stimulate both proliferation anddifferentiation (Pharr et al. Proc. Natl. Acad. Sci. USA 90, 938(1993)).

Activation of the EPO receptor may be due to its dimerization. That is,EPO may act as a crosslinker between two EPO receptor molecules. Thereis evidence in support of this proposal. An arginine to cysteinemutation at position 129 of the murine EPO receptor results inconstitutive activation of the receptor, presumably because of adisulfide bond formed between two receptors subunits (Yoshimura et al.Nature 348, 647 (1990)). In addition EPOR is found in multimericcomplexes in cells (Miura & Ihle Arch. Biochem. Biophys. 306, 200(1993)). However, isolation of a stable multimeric form of purified EPOsoluble receptor has not been reported. In addition, dimerization ofEPOR may be required, but not by itself be sufficient for completeactivation of cells. For example, dimerization may result in aproliferative signal but not a differentiation signal. That is,accessory proteins may be required to send the differentiation signal.

The possible relationship between EPO receptor dimerization andactivation may be exploited to identify compounds which are differentfrom EPO but activate the receptor. For example, antibodies possess twoidentical binding sites for antigen. An anti-EPOR antibody can bind twoEPOR molecules and could bring them into close proximity to each otherto allow dimerization. In order to function in vivo, these antibodiesmust recognize the EPOR on surfaces of cells and bind in a way thatallows activation of the signal transduction pathway. In addition, it isdesirable that activation result in both proliferation anddifferentiation of erythroid progenitors. A similar approach tounderstand the activation of human growth hormone receptor (Fuh et al.Science 256, 1677 (1992)) and epidermal growth factor receptor(Schreiber et al. Proc. Natl. Acad. Sci. USA 78, 7535 (1981)) has beenreported

It would be desirable to identify molecules which have the property ofactivating the EPO receptor and stimulating erythropoiesis. In order todo so, an understanding of the mechanism of EPO receptor activation andsignal transduction is important. One approach to elucidating thismechanism may be to identify antibodies which recognize the EPO receptorso as to activate the receptor and stimulate erythropoiesis. Suchantibodies are useful in therapeutic and diagnostic applications andwould also be useful for probing EPO receptor function.

The following references describe antibodies which bind to the mouse orhuman EPO receptor:

D'Andrea et al. in The Biology of Hemtaopoiesis, Wiley-Liss, Inc. (1990)pp. 153-159, generated polyclonal anti-peptide antibodies against anamino-terminal and a carboxy-terminal peptide of murine EPO receptor.The antibodies were shown to react with mouse EPO receptor in a Westernblot.

Bailey et al. Exp. Hematol. 21, 1535-1543 (1993) generated polyclonalanti-peptide antibodies against synthetic peptides homologous to theextraceullular and cytoplasmic domains of the mouse EPO receptor.Receptor activation by these antibodies, as measured by 3H thymidineuptake into spleen cells from phenylhydrazine treated mice, was notdetected.

Baynes et al. Blood 82, 2088-2095 (1993) generated a polyclonal antibodyto an amino-terminal peptide in the human EPO receptor. The antibody wasshown to react with a soluble form of the receptor present in humanserum.

D'Andrea et al. Blood 82, 46-52 (1993) generated monoclonal antibodiesto human EPO receptor. The antibodies bind to Ba/F3 cells transfectedwith the human EPO cDNA clone and some inhibit EPO binding and neutalizeEPO-dependent growth.

Fisher et al. Blood 82, 197A (1993) used the same monoclonal antibodiesas described in D'Andrea, supra to distinguish erythroid progenitorcells having EPO-dependent growth and maturation from those havingEPO-independent growth and maturation.

None of the antibodies described in the aforementioned references werereported to activate the EPO receptor or stimulate the growth and/ormaturation of erythroid progenitor cells.

Therefore, it is an object of the invention to produce antibodies whichrecognize an EPO receptor and bind to it such that the receptor isactivated. It is a further object of the invention to produce antibodieswhich bind to an EPO receptor and stimulate erythropoiesis bystimulating the proliferation and/or differentiation of erythroidprogenitor cells to erythrocytes. Such antibodies are useful in thetreatment of anemia or in the diagnosis of diseases characterized bydysfunctional EPO receptor. Further, such antibodies may lead to theidentification of therapeutic agents for the treatment of anemia.

SUMMARY OF THE INVENTION

The invention relates to antibodies or fragments thereof which activatean erythropoietin receptor. Screening of antibodies which recognize thehuman EPO receptor has revealed that two antibodies, designated Mab 71and Mab 73, stimulated the proliferation of UT7-EPO cells, anEPO-dependent cell line that does not proliferate in the absence ofadded EPO. Further, Mab 71 stimulated erythoid colony formation fromerythroid progenitors in human blood. The antibodies encompassed by theinvention may recognize an epitope on an EPO receptor which isrecognized by Mab 71 or Mab 73. The antibodies are preferably monoclonalantibodies and may be humanized or human antibodies. Also included arehybridoma cell lines which produce the antibodies of the invention.

Also provided for are methods and kits for detecting EPO receptors inbiological samples wherein the methods and kits comprise EPO receptorantibodies of the invention. Pharmaceutical compositions comprising EPOreceptor antibodies and pharmaceutically acceptable adjuvants are alsoencompassed by the invention. Such compositons may be used to treatpatients having disorders characterized by low red blood cell levels.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an ELISA assay that measured the binding tothe indicated concentrations of synthetic peptides by Mab 71. Thepeptides correspond to the indicated amino acid residues of human EPOreceptor. Residue 1 is the amino terminal proline found in secreted EPORupon cleavage of the leader sequence.

FIG. 2 shows the effect of varying amounts of rHuEPO protein andpurified Mabs 71 and 73 on ³H thymidine uptake of UT7-EPO cells.

FIG. 3 shows the effect of varying amounts of rHuEPO protein, Mab 71,Mab 73 or a non neutralizing control Mab directed against EPO (Mab F12)on inhibition of ¹²⁵I EPO binding to EPO receptors on the surface ofOCIM1 cells.

FIG. 4 shows a coomassie stained SDS gel of purified preparations ofmonoclonal antibodies 71 and 73 as well as monoclonal antibody fragments(Fabs) derived from Mabs 71 and 73. Samples were run under eitherreducing (plus 2-mercaptoethanol) or nonreducing (minus2-mercaptoethanol) conditions.

FIG. 5 shows the effect of varying amounts of purified rHuEPO protein,Mab 71 or Fab 71 on ³H thymidine uptake of UT7-EPO cells.

FIG. 6 shows the effect of varying amounts of purified Mab 71 or Fab 71on ³H thymidine uptake of UT7-EPO cells to which are also added 30munits/ml of recombinant human EPO (rHuEPO).

FIG. 7 shows a photograph of purified CD 34⁺ cells from peripheral bloodwhich were grown 21 days in methylcellulose in the presence of EPO orMab 71 under serum free growth conditions. Photos are of cells incubatedwith 500 munits/ml EPO (A), 25 munits/ml EPO (B), or 2.1 micrograms/mlMab 71(C).

FIG. 8 shows the effect of varying amounts of rHuEPO, Mab 71 and acontrol monoclonal antibody raised to Her2/neu on the formation oferythroid colonies from erythroid precursors when grown under serum freegrowth conditions in soft agar.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibodies (Mabs) which recognize the erythropoietin receptorhave been generated by immunizing mice with purified soluble human EPOreceptor. Soluble human EPO receptor was expressed and purified asdescribed in Examples 1 and 2. Of those Mabs which reacted with solublehuman EPO receptor in enzyme-linked immunosorbent assays (ELISAs), 96mabs were selected for further screening. These mabs were tested for EPOreceptor binding by BIAcore analysis (Example 4A) and for binding to EPOreceptor on the surface of transfected CHO cells by FACS (Example 4C).The results of these screenings are shown in Table 1. While a number ofantibodies bound EPO receptor as determined by BIAcore analysis, onlyfive antibodies of the 96 tested bound EPO receptor displayed on thesurface of transfected CHO cells as determined by FACS scanning. 24antibodies which were positive in ELISA assays (including those fivewhich were positive by FACS scanning) were tested for stimulation ofUT7-EPO cell proliferation. Surprisingly, it was found that twoantibodies, designated Mab 71 and Mab 73, stimulated the uptake of 3Hthymidine into a UT7-EPO cell line (Komatsu et al. Blood 82, 456 (1993))in the absence of EPO (Example 8A). The UT7-EPO cell line requires thepresence of EPO in its medium for growth. Therefore, the stimulation ofUT7-EPO cell growth is likely due to the activation of EPO receptor byMab 71 and Mab 73. As shown in FIG. 2, the response of UT7-EPO cells wasgreater in the presence of Mab 71 than Mab 73. It was further found thatMab 71 stimulated erythroid colony formation from human erythroidprecursors (see Example 9). This is the first instance of an antibodystimulating the formation of erythroid colonies from erythroidprecursors.

The invention provides for an antibody or fragment thereof whichactivates an erythropoietin receptor. As used herein, the term“activation of an EPO receptor” denotes one or more molecular processeswhich an EPO receptor undergoes that result in transduction of a signalto the interior of a receptor-bearing cell, wherein the signalultimately brings about one or more changes in cellular physiology.Cellular responses to EPO receptor activation are typically changes inthe proliferation or differentation of receptor-bearing cells.Receptor-bearing cells are typically erythroid progenitor cells.Presently, the molecular events leading to signal transduction by EPOreceptor are poorly understood. However, as indicated in the background,some evidence suggests that EPO receptor dimerization is at least oneevent which is likely to be required for activation. The presentdisclosure also provides support for this idea. As shown in FIG. 5,stimulation of 3H-thymidine uptake in UT7-EPO cells by Mab 71 isabolished when substituted by the corresponding Fab fragment designatedFab 71. Therefore, replacement of the intact, bivalent antibody with acorresponding monovalent fragment eliminates the proliferative response.In addition Mab 71 inhibits activation of the EPO receptor at highconcentrations. Both of these observations support the dimerizationmodel of activation for the EPO receptor. Mab 71 has been shown tointeract with a synthetic peptide of residues 49 to 78 of the humanEPO-R (see example 6). Thus this region of EPO-R when bound by a crosslinker such as Mab 71 can result in activation of EPO-R. It isunderstood that molecules that cross-link two EPO-R molecules by bindingto residues 49 to 78 are also encompassed by the invention. Thesemolecules could be antibodies or other bivalent molecular entities thathave the property of crosslinking two EPO receptors by binding toresidues contained within the region between residues 49 and 78 therebyresulting in dimerization and activation of the EPO receptor.

EPO receptors of the invention will preferably be mammalian EPOreceptors and, in a particularly preferred embodiment, will be human EPOreceptor. It is understood that analogs of human EPO receptors are alsoencompassed by the invention. Such analogs are constructed byinsertions, deletions, extensions or substitutions of amino acids in thehuman EPO receptor sequence. Examples of EPO-R analogs have beendescribed in U.S. Pat. No. 5,292,654 to Yoshimura et al. whereinsubstitution of a cysteine residue at position 129 of the EPOR aminoacid sequence resulted in constitutively activated EPOR. In general,EPO-R analogs having amino acids changes in regions other than theantibody binding domains necessary for activation wherein said analogsretain secondary and tertiary structure of the human EPO receptor may berecognized by the antibodies of the present invention. It has been shownthat Mab 71 interacts with a synthetic peptide of residues 49 to 78 ofthe human EPO-R (see Example 6). Therefore, EPO-R analogs having changesin amino acid residues other than those at positions 49 to 78 andretaining the human EPO receptor secondary and tertiary structure arelikely to be recognized by Mab 71. The numbering of amino acid residuesin the human EPOR polypeptide as used herein starts with proline atposition 1, which is the amino terminal residue after cleavage of the 25amino acid signal peptide.

Antibodies of the invention bind to an epitope on an EPO receptor whichis involved in receptor activation. In one embodiment, antibodiesrecognize an epitope on an EPO receptor which is recognized by Mab 71 oran epitope which is recognized by Mab 73. Mab 71 recognizes a syntheticpeptide spanning amino acid residues 49 to 78 in the human EPO-R.Therefore, it is likely that Mab 71 recognizes an epitope on EPO-R whichis defined in whole or in part by this sequence. As used herein, theterm “epitope” refers to the region of an EPO-R bound by an antibodywherein the binding prevents association of a second antibody to anEPO-R.

The invention also provides polyclonal antibodies, and monoclonalantibodies and fragments thereof. Antibody fragments encompass thosefragments which activate an EPO receptor. Also encompassed are humanizedantibodies, typically produced by recombinant methods, wherein humansequences comprise part or all of an antibody which activates an EPOreceptor. Examples of humanized antibodies include chimeric orCDR-grafted antibodies (U.S. Pat. Nos. 4,816,567 and 5,225,539). Alsoincluded are fully human antibodies to EPO receptor produced ingenetically-altered mice (see PCT Application No.93/12227). Antibodiesof the invention may also have a detectable label attached thereto. Sucha label may be a fluorescent (e.g., fluorescein isothiocyanate, FITC),enzymatic (e.g, horseradish peroxidase), affinity (e.g., biotin) orisotopic label (e.g., ¹²⁵I).

Also encompassed by the invention are hybridoma cell lines producing amonoclonal antibody which activates an EPO receptor. In one embodiment,the hybridoma cell line produces a monoclonal antibody which recognizesan eptitope on an EPO receptor which is recognized by Mab 71 or Mab 73.Generation of hybridoma cell lines producing monoclonal antibodies tohuman EPO-R are described in Example 3. The hybridoma cell line whichproduces Mab 71 has been deposited with the American Type CultureCollection, Rockville, Md. on ______ under accession no. ______. Thehybridoma cell line which produces Mab 73 has been deposited with theAmerican Type Culture Collection, Rockville, Md. on ______ underaccession no. ______.

The antibodies of the present invention are useful in diagnosing anemiaand other diseases characterized by dysfunctional EPO-R. In oneembodiment, a method of detecting in a biological sample an EPO receptorwhich is capable of which being activated comprising the steps of: (a)contacting the sample with an antibody which activates an EPO receptor;and (b) detecting activation of the receptor by the antibody. Thebiological samples include tissue specimens, intact cells, or extractsthereof. Antibodies may be used as part of a diagnostic kit to detectthe presence of EPO receptors in a biological sample. Such kits employantibodies having an attached label to allow for detection. Theantibodies are useful for identifying normal or abnormal receptors. Thepresence of abnormal receptors in a biological sample may be indicativeof disorders such as Diamond Blackfan anemia, where it is believed thatthe EPO receptor is dysfunctional.

Antibodies of the invention are useful for treating disorderscharaterized by low red blood cell levels. Included in the invention aremethods of modulating the endogenous activity of an EPO receptor in amammal, preferably methods of increasing the activity of an EPOreceptor. In general, any condition treatable by erythropoietin, such asanemia, may also be treated by the antibodies of the invention.Therapeutic antibodies are administered by an amount and route ofdelivery that is appropriate for the nature and severity of thecondition being treated and may be ascertained by one skilled in theart. Preferably, administration is by injection, either subcutaneous,intramuscular, or intravenous.

The invention provides for a pharmaceutical composition comprising atherapeutically effective amount of an antibody which activates an EPO-Rtogether with a pharmaceutically acceptable adjuvant, wherein theadjuvant may be selected from one or more of a diluent, carrier,preservative, emulsifier, anti-oxidant and/or stabilizer. A“therapeutically effective amount” as used herein refers to that amountof antibody which provides a therapeutic effect for a given conditionand administration regimen. In the present invention, the therapeuticeffect is stimulation of red blood cell production as evidenced by arise in hematocrit in the patient being treated. In a preferredembodiment, the antibodies are humanized or human antibodies which maybe prepared using procedures known to the skilled worker.Pharmaceutically acceptable adjuvants are known to one skilled in theart and are surveyed extensively in Remington's Pharmaceutical Sciences,18th ed. A. R. Gennaro, ed. Mack, Easton, Pa. (1990).

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

EXAMPLE 1 Production of Soluble Human Erythropoietin Receptor

A. Isolation of Clones for Expression of Soluble Human ErythropoietinReceptor.

Using a clone containing the human erythropoietin receptor as describedby Jones et al. supra, the PCR technique was used to obtain a clone forexpression of soluble human erythropoietin receptor (sHuEPOR). Primersfor PCR amplification of human erthropoietin receptor were: 5′ primer:CTC CAA GCT TGC CGT CAC CAT GGA CCA (SEQ. ID NO:_) CCT CGG GGC GTC CCT;and 3′ primer: CAG GTC TAG ATT ACT AGG GAT CCA GGT (SEQ. ID NO:_) CGCTAG GC

PCR reactions were carried out using 2.5 ng of a plasmid containinghuman EPOR, 5 pmol of each of the above oligonucleotide primers, 10 mMTris HCl (pH 8.3), 50 mM KCl, 1.5 mM Mg Cl₂, 200 μM each dNTP and 1 unitof Taq polymerase. Amplification was for 5 cycles of 30 sec. at 94° C.,1 min. at 50° C., 1 min at 72° C., followed by 20 cycles of 30 sec. at94° C., 1 min. at 55° C., 1 min at 72° C. DNA was purified by passagethrough a G-50 size exclusion column (Boehringer Mannheim Corp.), thendigested with Hind III and XbaI and ligated into the expression vectorpDSRα2 (DeClerck et al. J. Biol. Chem. 26, 3893 (1991)) which has alsobeen digested with Hind III and XbaI. Clones containing the desiredinsert were verified by DNA sequence analysis.

The d40EPOR clone was made by PCR from a full length human EPOR clone(see above). The carboxy terminus of d40EPOR is tyr467, the result ofadding a stop codon within the primer. Primers for PCR amplificationwere: 5′ primer: 5′-CTC CAA GCT TGC CGT CAC CAT GGA (SEQ. ID NO:_) CCACCT CGG GGC GTC CCT-3′; and 3′ primer: 5′-AGG TCG ACT ACT AGT AGT CAGTTG (SEQ. ID NO:_) AGA-3′PCR amplification used pfu polymerase in pfu buffer2 (Stratagene, LaJolla, Calif.). Reaction conditions were: 1 cycle at 96° for 30 sec.,45° for 1 min., 72° for 1 min.; 25 cycles at 96° for 1 min., 55° for 1min., 72° for 2 min. A final 720 incubation for 5 min. was thenperformed. The reaction products were separated by agarose gelelectrophoresis and the approximately 1.3 Kb band was isolated using agene clean kit (BIO 101, Vista, Calif.). The purified fragment wasligated into PCR II (TA cloning kit, Invitrogen, San Diego, Calif.).Recombinants were identified by restriction analysis and sequenced toconfirm the desired inserts were present. A HindIII-Sall fragment wasisolated as described above and ligated into an isolated pDSRo2 vectorthat had been previously cut with HindIII and SalI. The resultantvector, pDSRαEPORd40 was used for expression in CHO cells.B. Expression of Soluble Human EPOR and d40 EPOR in CHO Cells

The expression plasmid pDSRα2-EPOR-X contains sequences encoding humanEPOR amino acids Met1-Pro249 as shown in Jones et al. supra. PlasmidpDSRαEPORd40 contains sequences encoding Met1-Tyr467. Ten micrograms ofeach plasmid were independently introduced into CHO cells by calciumphosphate mediated transfection (Wigler et al. Cell 11, 233 (1977)).Individual colonies were selected based upon expression of thedihydrofolate reductase gene from the vector. Expression of human EPORwas monitored by RNA hybridization (Hunt et al., Exp. Hematol, 19: 779(1991)) and by Western immuno blotting using an affinity purifiedantibody. Cell lines which were positive in these assays were selectedfor further expansion. Cell lines were adapted to 30 nM Methotrexate(Mtx) to stimulate amplification of EPO-R expression.

Generation of conditioned media containing soluble human EPOR was donein both roller bottles and a hollow fiber bioreactor. Roller bottleswere innoculated with 2×10⁷ cells in 200 ml growth medium (DMEM: Ham'sF12 (1:1) supplemented with non-essential amino acids (NEAA), 30 nM Mtxand 5% fetal bovine serum (FBS) (reagents from GIBCO, Grand Island,N.Y.)). Upon reaching confluence in 3-4 days, the media was replacedwith 200 ml DMEM: Ham's F12, NEAA, 30 nM Mtx with no serum. Conditionedmedia was harvested after 6-7 days and replaced with fresh serum-freemedia. Second and third harvests were collected.

A Cell Pharm biorector cartridge was innoculated with 5×10⁸ cells ingrowth medium (as above) supplemented with 5 ug/mL gentamicin. The pHwas maintained at 7.3. Beginning on day 12 after innoculation the cellswere weaned off of serum to generate serum-free conditioned media.Harvesting of conditioned media began on day 17.

EXAMPLE 2 Purification of Soluble Human Erythropoeitin Receptor

Four different preparations of soluble recombinant human EPOR were made.In the first preparation, Epoxy-activated Sepharose 6B (Pharmacia,Piscataway, N.J.) is coupled with recombinant human erythropoietin(rHuEPO) essentially as per manufacturer's instructions. 218 mg ofrHuEPO in 4.5 mL of 32 mM ZnCl₂ is added to 7.2 g of Epoxy-activatedSepharose 6 B previously hydrated and washed with H₂O. This slurry istitrated to pH 10.8 then mixed overnight at room tempurature. Anyremaining reactive groups are then blocked by addition of ethanolamineto a final concentration of 1 M and mixed for 4 hours at roomtemperature. The subsequent steps are performed at 8°±2° C. The coupledresin (Epoxy-EPO) is packed into a column and washed with alternatingcycles of 0.5 M NaCl/0.1 M HOAc pH 4 and 0.5 M NaCl/0.1 M Borate pH 8.The column is equilibrated with 140 mM NaCl/10 mM Tris pH 7.6 (TBS). Itis loaded with 1560 mL of roller bottle produced conditioned media fromCHO cells expressing soluble EPO-R (sHuEPO-R). After loading iscomplete, the column is washed with 300 mM NaCl/10 mM Tris pH 7.6 thenthe bound sHuEPOR is eluted with 1× NaCl/3 M urea/10 mM Tris pH 7.6. TwoUV₂₈₀ absorbing peaks elute with this buffer. The second peak to elute,which contains the sHuEPOR, is pooled and diluted 20 fold with H₂O. Thediluted pool is then loaded to a 1 mL prepacked column of Mono Q(Pharmacia) and eluted with a NaCl gradient in 10 mM Tris pH 7.6. Asingle peak elutes, which is pooled, aliquoted and stored frozen at −80°C.

In the second preparation, a larger Epoxy-EPO column is made. 20.4 g ofEpoxy-activated Sepharose 6 B is hydrated and washed with H₂O, then withacetone and finally with 50% formamide in H₂O pH 10.6. 729 mg of rHuEPOin 15 mL of H₂O is titrated to pH 10.6, added to the resin and mixedovernight at room tempurature. Any remaining reactive groups are thenblocked by addition of ethanolamine to a final concentration of 1× andmixed for 140 minutes at room temperature. The subsequent steps areperformed at 8°±2° C. The Epoxy-EPO is packed into a column and washedwith 3 M urea/750 mM NaCl/10 m Tris pH 7.6, the column is thenequilibrated with TBS. 100 mL of bioreactor produced conditioned mediafrom CHO cells expressing sHuEPOR are mixed with 2 mL of Q SepharoseFast Flow (Pharmacia). It is incubated for 30 minutes at 8°±2° C. withfrequent mixing, then filtered through a 0.45 micron cellulose nitratebottle top filter (Corning). The filtrate is loaded to the Epoxy-EPOcolumn, washed with 250 mM NaCl/10 mM Tris pH 7.6, then eluted with 3 Murea/750 mM NaCl/10 mM Tris pH 7.6. The eluted peak is pooled anddiluted 20 fold with H₂O. The diluted pool is then loaded to a 15 mLcolumn of Q Sepharose Fast Flow and eluted with a NaCl gradient in 10 mMTris pH 7.6. The single peak that elutes is pooled, aliquoted and storedfrozen at −80° C.

In the third preparation, the same Epoxy-EPO column used in preparation2 is used. 850 mL of roller bottle produced conditioned media from CHOcells expressing sEPO-R are mixed with 1.7 mL of Q Sepharose Fast Flow.It is processed in the same manner as is done in preparation 2.

In the fourth preparation, 7.25 L of bioreactor produced conditionedmedia from CHO cells expressing sHuEPOR are mixed with 110 mL of QSepharose Fast Flow. It is incubated for 1 hour at 8°±2° C. withfrequent mixing, then filtered through a 0.45 micron cellulose nitratebottle top filter. The filtrate is then diluted with 7.25 L of H₂O andloaded to a 770 mL column of Q Sepharose Fast Flow equilibrated in 20 mMTris pH 7.6. The column is eluted with a NaCl gradient in 20 mM Tris pH7.6. Fractions containing significant amounts of sHuEPOR based onSDS-PAGE analysis are pooled. Solid (NH₄)₂SO₄ is added to the pool to afinal concentration of 1.2 M then filtered through a 0.45 microncellulose nitrate bottle top filter The filtrate is loaded to a 60 mLcolumn of Phenyl Sepharose 6 (low sub, Pharmacia) and eluted with adecreasing gradient of 1.2 M to 0 M (NH₄)₂SO₄ in 20 mM Tris pH 7.6. Themajor eluting peak is pooled and made 2.4 M in (NH₄)₂SO₄ to precipitatethe sHuEPORt. The precipitated sHuEPOR is harvested by centrifugation,resuspended with H₂O and titrated to pH 7.9 with Tris-HCl. The resultantsolution is filtered through a 0.45 micron cellulose nitrate filter,aliquoted and stored frozen at −80° C.

EXAMPLE 3 Preparation and Screening of Hybridoma Cell Lines

A. Enzyme-Linked Immunosorbent Assay (EIA)

EIAs were initially performed to determine serum antibody (Ab) titres ofindividual animals, and later for screening of potential hybridomas.Flat bottom, high-binding, 96-well microtitration EIA/RIA plates (CostarCorporation, Cambridge, Mass.) were coated with purified sHuEPOR at 5 μgper ml carbonate-bicarbonate buffer, pH 9.2 (0.015 M Na₂CO₃, 0.035 MNaHCO₃). Fifty μl of the Ab were added to each well. Plates were thencovered with acetate film (ICN Biomedicals, Inc., Costa Mesa, Calif.)and were incubated at room temperature (RT) on a rocking platform for 2hours or over-night at 4° C. sHuEPOR lot #1 was used after the first andsecond boost, lot #2 was used after the third boost. sHuEPOR lots #3 and4 were used for screening of hybridomas. Plates were blocked for 30minutes at RT with 250 μl per well 5% BSA solution prepared by mixing 1part BSA diluent/blocking solution concentrate (Kirkegaard and PerryLaboratories, Inc.) with 1 part deionized water (dH₂O). Blockingsolution having been discarded, 50 μl of serum 2-fold dilutions (1:400through 1:51,200) or hybridoma tissue culture supernatants were added toeach well. Serum diluent was 1% BSA (10% BSA diluent/blocking solutionconcentrate diluted 1:10 in Dulbecco's Phosphate Buffered Saline, D-PBS;Gibco BRL, Grand Island, N.Y.), while hybridoma supernatants were testedundiluted. In the case of hybridoma testing, one well was maintained asa conjugate control, and a second well as a positive Ab control. Plateswere again incubated at RT, rocking, for 1 hour, then washed 4 timesusing a 1× preparation of wash solution 20× concentrate (Kirkegaard andPerry Laboratories, Inc.) in dH₂O. Goat anti-mouse IgG heavy- andlight-chain specific horseradish peroxidase conjugated secondary Ab(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) diluted 1:1000 in1% BSA was then incubated in each well for 30 minutes. Plates werewashed as before, blotted dry and ABTS Peroxidase single componentsubstrate (Kirkegaard and Perry Laboratories, Inc.) was added.Absorbance was read at 405 nm for each well using a Microplate EL310reader (Bio-tek Instruments, Inc., Winooski, Vt.). Half-maximal titre ofserum antibody was calculated by plotting the log₁₀ of the serumdilution versus the optical density at 405 nm, then extrapolating at the50% point of the maximal optical density obtained by that serum.Hybridomas were selected as positive if optical density scored greaterthan 5-fold above background.

B. Immunization

Ten, 4.5 week old Balb/c mice (Charles Rivers Laboratories, Wilmington,Mass.) were subcutaneously injected (SQI) with 50 μg sHuEPOR; lot 1;antigen) emulsified in Complete Freund's Adjuvant (CFA; 50% vol/vol;Difco Laboratories, Detroit, Mich.). These animals were boosted (SQI) 4weeks later with 25 μg antigen (Ag; lot 1) prepared in similar fashionusing Incomplete Freund's Adjuvant (ICFA; Difco Laboratories, Detroit,Mich.). Mice were bled via the tail 9 days later and serum antibody (Ab)titres determined by enzyme-linked immunosorbent assay (EIA). As the ½maximal titre for each mouse rose above 5000, individual animals wereselected for the hybridoma preparation. The three animals (#7, 8 and 9)which were used to generate the hybrids of interest (#71A and 73A)required additional boosts at 5 weeks and again at 29 weeks using 12.5μg Ag (lot 1) and 25 μg Ag (lot 2) respectively. These boosts wereperformed in the same manner as the initial boost; that is, as anemulsion in 50% vol/vol ICFA. Serum Ab titres continued to be monitored9 days following each boost. The final titres of these mice prior tofusion were 5026, 6842, and 12,945 for animals 7, 8 and 9, respectively.

C. Cell Fusion

Animals 7, 8 and 9 were intravenously injected with 25 μg of sHuEPOR(lot #3) 8 weeks following the final boost. Four days later, mice weresacrificed by carbon dioxide and spleens collected under sterileconditions into 25 ml Dulbecco's Modified Eagle's Medium containing 200U/ml Penicillin G, 200 μg/ml Streptomycin sulfate, and 4 mM glutamine(2× P/S/G DMEM). The spleens were trimmed of excess fatty tissue, thenrinsed through 3 dishes of clean 2× P/S/G DMEM. They were nexttransferred to a sterile stomacher bag (Tekmar, Cincinnati, Ohio)containing 10 ml of 2× P/S/G DMEM, and disrupted to single cellsuspension with the Stomacher Lab Blender 80 (Seward Laboratory UACHouse; London, England). As cells were released from the spleen capsuleinto the media, they were removed from the bag and passed through a 70μm nylon mesh cell strainer (Becton Dickinson and Company; Lincoln Park,N.J.). Fresh media was replaced in the bag and the process continueduntil the entire cell content of the spleens were released. Thesesplenocytes were washed 3 times by centrifugation at 225×g for 10minutes. In the first fusion, splenocytes from animal #9 were used; inthe second fusion, splenocytes from animals #7 and 8 were pooled.

Concurrently, log phase cultures of Sp2/0-Ag14 mouse myeloma cells(available from the American Type Culture Collection, Rockville, Md.under accession no. CRL 1581) grown in complete medium (DMEM, 10% fetalbovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mMsodium pyruvate, and 10 mM Hepes Buffer; Gibco Laboratories, Inc., GrandIsland, N.Y.), were washed in similar fashion. From this myelomapopulation, 4×10⁷ cells (fusion 1) or 8×10⁷ cells (fusion 2) were taken,mixed with the suspension of splenocytes, and pelleted once again. Themedia was aspirated from the cell pellet and 2 ml of polyethylene glycol(PEG 1500 MWt; Boehringer Mannheim Biochemicals, Indianapolis, Ind.) forfusion 1 of 3.5 ml of PEG for fusion 2 at 37° C. were gently mixed intothe media over the course of 1 minute. Thereafter, an equal volume of 2×P/S/G DMEM was slowly added. The cells were allowed to rest at 37° C.for 2 minutes, then an additional 9 ml of 2× P/S/G DMEM added. The cellswere again set at 37° C. for 4 minutes. Finally, 30 ml of 2× P/S/G DMEMwas added to the cell suspension, and the cells pelleted bycentrifugation. Media was aspirated from the pellet and the cells gentlyresuspended into approximately 56 ml (fusion 1) or 74 ml (fusion 2) ofcomplete medium containing 100 U/ml Penicillin G and 100 μg/mlStreptomycin Sulfate. Cells were distributed over 10 96-well flat bottomtissue culture plates (Becton Dickinson Labware; Lincoln Park, N.J.) bysingle drops from a 5 ml pipette. Plates were incubated in humidifiedconditions at 37° C., 5% CO₂, overnight. The next day, an equal volumeof selection medium was added to each well. Selection consisted of 0.1mM hypoxanthine, 4×10⁻⁴ mM aminopterin, and 1.6×10⁻² mM thymidine incomplete medium. The fusion plates were incubated for 7 to 10 days with2 changes of medium during this time; HAT selection medium was usedafter each fluid change. Tissue culture supernatants were taken fromeach hybrid-containing well and tested by EIA for specific antibodyreactivity to sHuEPOR. 96 wells which were positive in EIA weresubjected to further screening.

D. Dot Blots

Dot blots of reduced sHuEPOR (lot #4) were used as a secondary screeningmethod for EIA positive hybridomas. The Dot Blot SF MicrotitrationApparatus (Bio-Rad Laboratories, Inc.; Richmond, Calif.) was set-upaccording to the instruction manual; nitrocellulose membranes (9×12 cm;Bio-Rad Laboratories, Inc.; Richmond, Calif.) were employed. Antigen wasfirst prepared by boiling for 5 minutes under reducing conditions with2-mercaptoethanol (5% vol/vol; Bio-Rad Laboratories, Inc.; Richmond,Calif.) in Tris-buffered saline solution (TBS; 10 mM Tris pH 7.5, 154 mMNaCl, 0.01% wt/vol Na azide). Twenty-five ng of sHuEPOR (lot #4) wasloaded into each well and aspirated through the nitrocellulose membranefor binding. The wells were filled with 250 μl Blotto-Tween solution(block solution; 2% wt/vol non-fat dry milk, 50 mM Tris, pH 7.5, 25 mMNaCl, 0.1 mM EDTA, 0.09% vol/vol Tween 20, 0.01% vol/vol anti-foam A)and incubated at RT for 30 minutes. Block solution was aspirated fromthe wells and the procedure repeated for a second time to ensurecomplete blocking of non-specific sites on the membrane. This wasfollowed by 3 washes through the membrane with D-PBS containing 0.1%vol/vol polyoxyethylene sorbitan monolaurate (Tween-20; Bio-RadLaboratories, Inc.; Richmond, Calif.). Ninety-five μl of EIA-positivehybridoma conditioned medium was next added to each well and incubatedfor 45 minutes at RT. Wells were washed 3× with TBS-Tween (20 mM Tris,pH 7.5, 50 mM NaCl, 0.02% vol/vol Tween 20) and 2× with TBS-Tween (20 mMTris, pH 7.5 0.5 M NaCl, 0.09% vol/vol Tween 20) at 250 μl per wash,aspirating through the membrane after each addition. One-hundred μl ofgoat anti-mouse IgG, heavy- and light-chain specific, HRP-conjugatedsecondary antibody (1:1000 diluted in TBS-Tween; Boehringer MannheimBiochemicals; Indianapolis, Ind.) was incubated in each well for 45 minat RT. Membranes were washed as before, removed from the blot apparatus,dipped into prepared Enhanced Chemiluminescent Reagent (ECL reagent;Amersham Life Sciences, Corporation; Arlington Heights, Ill.), andexposed to X-OMAT AR film (Kodak Scientific Imaging, Rochester, N.Y.).Fifteen seconds later, the film was removed from film cassettes anddeveloped. Each well was scored 3+ to 0 based on intensity of dots forindividual hybridoma supernatants.

EXAMPLE 4 Anti-EPOR Antibody Binding to EPOR

A. Antibody Binding to EPO-R by BIAcore Analysis

Real-time biospecific interaction analysis (BIA, Pharmacia Biosensor AB,Uppsala, Sweden) based on surface plasmon resonance (SPR) (Fiagerstam etal. J. Mol. Recognition 3, 208 (1990); Malmbory et al. Scand. J.Immunol. 35, 643 (1992)) was used to screen the ELISA positivemonoclonal antibodies.

Soluble HuEPOR prepared as described in Examples 1 and 2 was covalentlycoupled to the sensor chip CM5 via the primary amine group. Theimmobilization was performed at a flow of 5 ul/min in HBS (10 mM HEPESpH7.4, 150 mM NaCl,3.4 mM EDTA, 0.05% BIAcore surfactant P-20). Thecarboxylated matrix of the sensor chip was first activated with a 40 ulinjection of 1:1 mixture of EDC (400 mMN-ethyl-N-(dimethylamine-propyl)carbodiimide in water, PharmaciaBiosensor AB) and NHS (100 mM N-hydroxysuccinimide in water, PharmaciaBiosensor AB). 65 ul of soluble EPO-R(50 ug/ml in 10 mM Na-acetatepH4.0) was injected to immobilize onto the sensor chip. The excessreactive groups of the sensor chip were deactivated with an injection of50 ul of ethanolamine (Pharmacia Biosensor AB)

Each analysis cycle included an injection of 20 ul of hybridomasupernatant, followed by injection of 10 ul of 10 mM HCl forregeneration of the chip. The SPR response is measured in ResonanceUnits (RU). For most proteins, 1000 RU corresponds to a surfaceconcentration of approximately 1 ng/mm². Results of screening 96 wellswhich were positive in EIAs are shown in Table 1. In these experiments,background is typically about 20 RU. Binding to EPOR is significant at50 RU and above. TABLE 1 EPO-R Monoclonal Antibodies BIACORE (3) FACS(4) Inhibition Stimulation ANTIBODY BIACORE COMPETITION MEAN of EPO ofUT7-EPO (1) (2) GROUP FLOURESCENCE Activity (5) Cells (6)  1 98 A — − − 2 8 NT — NT NT  3 7 NT — NT NT  4 65 NT — NT NT  5 13 NT — NT NT  6 9NT — − −  7 89 C — NT NT  8 46 NT — NT NT  9 29 NT — NT NT 10 69 NT — NTNT 11 4 NT — NT NT 12 153 C — NT NT 13 1499 B — NT NT 14 87 NT — NT NT15 29 NT — NT NT 16 8 NT — NT NT 17 7 NT — NT NT 18 46 NT — − − 19 9 NT— NT NT 20 7 NT — NT NT 21 49 NT — NT NT 22 8 NT — NT NT 23 4 NT — − −24 26 NT — NT NT 25 8 NT — NT NT 26 84 NT — NT NT 27 2 NT — NT NT 28 11NT — NT NT 29 1 NT — NT NT 30 270 A — − − 31 16 NT — − NT 32 18 NT — NTNT 33 15 NT — NT NT 34 25 NT — NT NT 35 363 A — NT NT 36 4 NT — NT NT 3716 NT — − − 38 13 NT — NT NT 39 574 B — − − 40 15 NT — NT NT 41 22 NT —NT NT 42 23 NT — NT NT 43 6 NT — NT NT 44 13 NT — NT NT 45 13 NT — NT NT46 7 NT — NT NT 47 10 NT — NT NT 48 5 NT — NT NT 49 69 NT — NT NT 50 345C — − − 51 31 NT — NT NT 52 6 NT — NT NT 53 130 A — NT NT 54 13 NT — NTNT 55 34 NT — NT NT 56 11 NT — NT NT 57 10 NT — NT NT 58 15 NT 14.99 + ?59 10 NT — NT NT 60 10 NT — NT NT 61 48 NT — NT NT 62 814 A — − − 631539 B — NT NT 64 1222 C — NT NT 65 −5 NT — +/− ? 66 975 C — NT NT 671000 A — − ? 68 495 C — NT NT 69 877 A — − − 70 789 A — − ? 71 1584 C23.55 +(7) +++ 72 1190 B — − − 73 354 C 13.71 − + 74 408 A 18.53 − − 75947 B — NT NT 76 6 NT — NT NT 77 434 C — − − 78 119 A — NT NT 79 8 NT —NT NT 80 11 NT — NT NT 81 −4 NT — NT NT 82 4 NT — NT NT   82B −13 NT NTNT NT 83 1025 C — − − 84 5 NT — NT NT 85 11 NT — NT NT 86 859 C — NT NT87 4 NT 12.81 − − 88 4 NT — +/− − 89 −1 NT — +/− − 90 4 NT — NT NT 91 0NT — − − 92 −3 NT — NT NT 93 2 NT — NT NT 94 5 NT — NT NT 95 417 A — NTNT 96 7 NT — NT NTTissue culture medium conditioned by hybridomas secreting the indicatedantibodies were tested with the assays indicated. Supernatantscontaining all the antibodies shown gave a positive signal in ELISAassays.+++, ++, + indicate a positive response with +++ indicating those havingthe greatest effect.− indicates a response less than or equal to the response of controlmedium.NT indicates samples were not tested.? indicates samples that could not be assigned a response.(1) Antibodies 1-61 are from mice number 7 and 8. Antibodies 62-96 arefrom mice number 9.(2) Response units by Mabs using biacore chip with attached sHuEPOR.(3) Competition on BIACORE was to anti sHuEPOR Mab 1G2. sHuEPOR bound toa sensor chip was incubated with 1G2 then effect on Mab binding comparedto binding to EPOR not preincubated with 1G2 was determined. Antibodieswhose binding was completely blocked (80-100%) are A. Antibodies whosebinding was blocked 50-80% are C. Antibodies whose binding was blockedless than 50% are B.(4) Values for antibodies that gave cells a mean fluorescence greaterthan the control (12.73) are shown “—” indicates antibodies with a meanfluorescence less that or equal to control.(5) Inhibition of 3H uptake by UT7-EPO cells. 30 munits of EPO andvarying amounts of antibody were incubated with cells. After anovernight incubation cells were pulse labeled with 3H thymidine and theamount of counts taken up were determined. A positive response wasdefined as one that had a progressive decrease with increasing amountsof antibody(6) Stimulation of 3H uptake by UT7-EPO cells. Varying amounts ofantibody were incubated with cells. After an overnight incubation, cellswere pulse labeled with 3H thymidine and the amount of counts taken upwere determined. A positive response was defined as one that had aprogressive increase in incorporation with increasing amounts ofantibody.(7) Inhibition was at concentrations higher than required to activate.B. Epitope Competition Analysis

The sensor chip which was immobilized with sHuEPOR could be saturated byan injection of 65 μl of hybridoma supernatant 1G2. 1G2 is a monoclonalantibody raised to sHuEPOR using procedures described in Example 3. Eachanalysis cycle included injections of 20 ul of the hybridoma supernatantwith and without one epitope being saturated by the injection of 65 ulof 1G2. The ratio of the binding signal in RU of 20 μl injection after1G2 saturation versus the binding signal in RU of 20 μl injection aloneis defined as % blocking by 1G2. Those antibodies with 80-100% blockingare assigned as group A, those with less than 50% blocking as group B,and those with 50-80% blocking as group C. The results are shown inTable 1.

C. Antibody Binding to d40EPOR on Transfected CHO Cells byFluorescence-Activated Cell Sorting (FACS) Analysis

Hybridoma supernatants raised against EPOR were tested for binding toEPO receptor on the surface of pDSRαEPORd40 transfected CHO cells byFACS analysis. CHO cells transfected with DNA encoding d40 EPO receptorwere constructed as described in Example 1. CHO/EPOR cells were scrapedfrom tissue culture dishes and resuspended as single cells in a solutionof PBS/0.5% BSA and were then distributed into a 96 well round-bottomplate at approximately 3×10⁵/well. The plate was then placed in thecentrifuge at 1000×g for 5 min. After centrifugation, the PBS/BSAsupernatant was removed and each of the pelleted cells were resuspendedin either a control media or in one of the EPOR hybridoma supernatants.The cells were incubated at 4° C. for 1 hour. After the incubation,cells were washed with PBS/BSA and then resuspended in a solution offluorescine isothiocyanate (FITC) labelled Goat anti Mouse monoclonalantibody (Southern Biotech, Birmingham Ala.). The cells were incubatedagain at 4° C. for 1 hour, washed and analyzed by FACS. Of the 96supernatants tested, five had a mean cell fluorescence greater thancontrol media (see Table 1). Mab 71 gave the highest level offluoresence followed by Mabs 74, 58, 73 and 87. No other supernatantstested exhibited fluorescence above control values.

EXAMPLE 5 Purification of Anti-EPOR Antibodies and Fab Fragments

A. Ascites Production

Balb/c mice (Charles Rivers Laboratories, Wilmington, Mass.), greaterthat 5 weeks of age were primed with 2,4,10,14-tetramethyl-pentadecane(Pristane; Sigma, St. Louis, Mo.) 7 to 10 days prior to injection ofcell lines. Each mouse received a single intraperitoneal injection of0.5 ml; 10 to 20 animals were injected for each cell line for whichascites fluid was to be prepared.

Hybridoma lines grown in complete medium until confluency was attained,were washed once with D-PBS then counted using a Neubauer Hemacytometer.Each mouse was then intraperitoneally injected with 10⁷ cells, andmaintained on Rodent Lab Chow and water ad libitum until ascites fluiddeveloped. Mice were monitored for maximum ascites formation, sacrificedunder CO₂, and tapped for fluid collection using an 18G needle insertedinto the fluid-filled cavity. The fluid was clarified by centrifugationat 225×g for 15 min or for 3 minutes in a microcentrifuge (Eppendorf).Four ml aliquots were then stored at −20° C. until purified by Protein-Acolumn chromatography.

B. Protein-A Purification of Monoclonal Antibodies:

Immunoglobulin from 4 ml of ascites fluid or 10 ml of hybridomaconditioned medium was purified by Protein-A column chromatography. TheBio-Rad Monoclonal Antibody Purification System II (MAPS II; Bio-RadLaboratories; Richmond, Calif.) was used. Briefly, 5 ml of Affi-gelProtein-A suspension was settled into a 1×10 cm disposable glass column.The Protein-A gel was washed with approximately 30 ml of D-PBS thenprepared by running 20 ml of Binding Buffer (MAPS II Binding Buffer;Bio-Rad) through the column. Ascites fluid or conditioned medium diluted1:1 with binding buffer was then added to the top of the column andallowed to flow through. After binding of immunoglobulin to Protein-A,the unbound fraction was discarded. The column was next rinsed ofunbound protein with 30 ml of binding buffer to yield an absorbance at280 nm of less than 0.01. The immunoglobulin-containing fraction wasthen eluted with Bio-Rad Elution buffer, approximately 30 ml. Thisfraction was buffer-exchanged overnight at 4° C. by dialysis against 4liters D-PBS. The resulting PBS-equilibrated immunoglobulin wasconcentrated by centrifugation at 1700×g in Centricon Concentrator units(Amicon Inc., Beverly, Mass.).

C. Fractionation of the Antibody-Binding Domain

Protein-A purified immunoglobulin was further fractionated into its 2component parts, the crystallizable fraction (Fc) and theantibody-binding fraction (Fab), using a Pierce ImmunoPure FabPreparation kit (Pierce Chemical Company, Rockford, Ill.). The protein-Apurified immunoglobulin was dialyzed into 20 mM phosphate/10 mM EDTAbuffer at pH 7.0, then concentrated to approximately 20 mg/ml. Ten mg ofimmunoglobulin was fractionated. Immobilized papain gel was rinsed twicewith digestion buffer containing 42 mg cysteine in 12 ml phosphatebuffer as supplied. The immunoglobulin sample was then added to the geland incubated at 37° C., on a rotating shaker, overnight. The solublizedFab was separated from the Fc and undigested immunoglobulin by protein-Apurification; unbound fraction was collected here as the Fab sample.This unbound portion was dialyzed overnight against 4 liters D-PBS at 4°C., and concentrated as before.

EXAMPLE 6 Mapping of Mab 71 Epitope on EPOR

Overlapping synthetic peptides 17 to 30 amino acids in length were madethat spanned residues 1 to 224 of the human EPO receptor, where residue1 is proline and residue 224 is aspartic acid. The ten differentpeptides overlapped by six amino acids at both ends. The sequences ofthe peptides and their location within the human EPO-R amino acidsequence are as follows: SE-1 PPPNLPDPKFESKAALLAARGPEELCFTE (residuses1-30) SE-2A LLCFTERLEDLVCFWEEA (residues 25-42) SE-2B CFWEEAASAGVGPGNYSF(residues 37-54) SE-3 PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (residues 49-78)SE-4 TARGAVRFWCSLPTADTSSFVPLELRVTAA (residues 73-102) SE-5LRVTAASGAPRYHRVIHINEVVLLDAPVGL (residues 97-126 SE-6DAPVGLVARLADESGHVVLRVLPPPETPMT (residues 121-150) SE-7PETPMTSHIRYEVDVSAGNGAGSVQRVEIL (residues 145-174) SE-8QRVEILEGRTECVLSNLRGRTRYTFAVRAR (residues 169-198) SE-9FAVRARMEAPSFGGFWSAWSEPVSLLTPSDLD (residues 193-224)

Polystyrene wells (Costar, Cambridge, Mass.) were coated with the aboveEPO-R peptides at concentrations of 100 μg/ml, 20 μg/ml and 0.8 μg/mlrespectively in carbonoate-biocarbonate buffer (0.015M Na₂C0₃, 0.035MNaHCO₃, pH 9.2). The plate was incubated at room temperature (RT) for 2hours then overnight at 4° C. Soluble HuEPOR was coated atconcentrations of 10 μg/ml, 2 μg/ml, 0.4 μg/ml and 0.08 μg/ml aspositive controls under the same conditions. After blocking with 5% BSAin PBS at RT for 30 minutes, the plate was incubated with Mab 71purified as described in Example 5 at a concentration of 5 μg/ml in 1%BSA at RT for 2 hours. After washing with washing buffer (Kirkegard andPerry Labs, Inc.) the plate was incubated with 1:1000 dilution of Goatanti-mouse IgG conjugated with horse Radish peroxidase (BoehringerMannheim) for one hour at RT. The plate was washed and developed withABTS (Kirkegard and Perry Labs, Inc.) substrate solution. Colorimetrywas conducted at 405 nm. The results of Mab binding to the syntheticpeptides are shown in FIG. 1 and indicate that Mab 71 binds significantamounts of peptide SE-3 (amino acid residues 49 to 78 inclusive of humanEPO-R) compared to the other peptides tested. This indicates that Mab 71binds to a region of the human EPO-R containing or overlapping residues49 to 78.

EXAMPLE 7 Activity of Anti-EPOR Antibodies in Cell Proliferation Assays

Antibodies in conditioned medium prepared as described above wereassayed for their ability to stimulate uptake of 3H-thymidine by UT7-EPOcells (Komatsu et al., supra). UT7-EPO cells are responsive to EPO andexpress human EPO receptors on their cell surface. UT7-EPO cells weregrown in Growth medium(1× Iscove's Modified Dulbecco's Medium withL-glutainine, 25 mM HEPES buffer, and 3024 mg/L sodium bicarbonate, butwithout either alpha-thioglycerol or beta-mercaptoethanol (GIBCO)/ 10%v/v Fetal Bovine Serum/1% v/v L-glutamine-Penicillin-Streptomycinsolution (Irvine Scientific)/1 Unit/ml rHuEPO) to approximately 3×10⁵cells/ml. Cells were collected by centrifugation (approx. 500×G) washedtwice with phosphate buffered saline and resuspended at 5×10⁴ cells/mlin Assay medium (1× RPMI Medium 1640 without L-glutamine (Gibco)/1%L-glutamine/4% fetal bovine serum). Test samples or EPO standard(rHuEPO), 100 μL diluted in assay medium at least 5-fold, were added towells in a 96 well microtiter plate. 50 μL cells were then added (5000cells/well) and plates were incubated in a humidified incubator at 37°C. and 5% CO₂. After 72 hours, 50μL methyl-³H-Thymidine (1 mCi/ml; 20Ci/mMole) diluted 1:100 in assay medium was added. Cells were incubatedfor an additional 4 hours at 37° C. and 5% CO₂. Labeled cells wereharvested onto glass fiber filtermats using a PHD cellharvester(Cambridge Technology Inc.) and deionized water as a washingsolution. Filters were rinsed a final time with 2-propanol then driedand counted in a Beckman Model LS6000IC scintillation counter.

Conditioned medium from tissue culture plates containing antiEPOR Mabswere tested for their ability to stimulate proliferation as describedabove. Samples at several dilutions were tested. Positive responses weredefined as those that stimulated thymidine uptake at least 2-fold overbackground levels and also resulted in decreasing stimulation as thesamples were diluted. As shown in Table 1, two samples out of 24 testedgave a positive response (Mabs 71 and 73). Four samples may have a weakstimulatory activity (? in Table 1). The remaining samples did not givea significant increase over background. A polyclonal serum from themouse used to generate monoclonals also stimulated thymidine uptake.This suggests that the polyclonal antibody in this serum was alsocapable of stimulating proliferation of UT7-EPO cells.

The supernatants were also tested for their ability to inhibitEPO-induced stimulation of thymidine uptake by UT7-EPO cells. Cells wereincubated with 25 munits/ml rHuEPO and varying amounts of antibodycontaining conditioned medium. Thymidine uptake was measured asdescribed above. The results are shown in Table 1. Most antibodies didnot significantly differ from control medium. Of the antibodies showinginhibition of thymidine uptake, two samples (Mabs 58 and 73) showeddefinite inhibition while three samples *(Mabs 65, 88 and 89) showedpossible inhibition. Mab 73 inhibited at the highest doses but at lowerdoses it stimulated thymidine uptake over control values.

EXAMPLE 8 Activation of EPOR by Anti-EPOR Antibodies and Fragments

A. UT7-EPO Proliferation Assay

Mabs 71 and 73 were purified as described in Example 5. Proliferativeactivity was determined with UT7-EPO thymidine uptake assays describedin Example 7. Both Mabs 71 and 73 stimulated uptake by UT7-EPO cells ina dose dependent manner as did rHuEPO (see FIG. 2). Activity was reducedat high doses of Mab 71. Peaks in stimulatory activity were observed atdoses of 1-2 μg/ml for Mab 71 and >100μg/ml for Mab 73. Anonneutralizing control antibody (AntiEPO Mab F12) did not stimulatewhich suggests that the stimulation is specific for EPO receptorantibodies.

B. EPO Cold Displacement Assays.

Antibodies to the EPO receptor may bind to the same region as EPO binds.To test this possibility, cold displacement assays were performed usingOCIM1 cells. OCIM1 cells are from human origin and known to contain EPOreceptors on their cell surface (Broudy et al. Proc. Nat. Acad. Sci. USA85, 6517 (1988)). Cells were grown in OCIM1 growth medium (Iscove'smodified Dulbecco medium(IMDM)/10% fetal bovine serum/1%pen-strep-fungisone) to approximately 2-5×10⁵ cells/ml. Cells werecollected by centrifugation, washed two times in binding buffer (RPMI1640/1% BSA/25 mM HEPES pH 7.3) then resuspended in binding buffercontaining 0.1% azide and 10 μg/ml cytochalisin Boat 1-2×10⁷ cells/ml.Cells (100 μL) in 96 well tissue culture plates were then incubated with10 μL sample and 10 μL ¹²⁵I-EPO (Amersham high specific activity; 3000Ci/mMole, 2 μCi/ml) in a 37° humidified tissue culture incubator. After3 hours cells were centrifuged through phthalate oil (60:40 (v/v)dibutyl/dinonyl phthalate) in titer tubes. The tubes containing cellswere quick frozen in a dry ice-ethanol bath and the cell pellet wasclipped and then counted in a LKB 1277 gammamaster automatic gammacounter.

FIG. 3 shows the results of the cold displacement experiment. Increasingamounts of ¹²⁵I-EPO were displaced from EPO receptors on cells as theamount of added unlabeled rHuEPO was increased. In a similar manner, Mab71 purified as described in Example 5 also displaced increasing amountsof ¹²⁵I-EPO with increasing amounts of antibody. In this case,approximately 4,000 fold more Mab 71 was needed than rHuEPO to displaceequivalent amounts of ¹²⁵I-EPO. In contrast Mab 73 showed indications ofdisplacement at the highest doses but a nonneutralizing anti rHuEPO Mab(F12) did not significantly displace. These results indicate that MabF12 did not interfere with binding of EPO to its receptor but Mab 71 and73 do. This result also indicates that Mab 71 binds to the EPO receptorand activates it by binding at or close to the EPO binding site.

C. Comparison of Activities of Mab 71 and Fab 71

EPO receptor fragments of Mab 71 were prepared as described in Example5. The preparations were characterized by SDS gel electrophoresis(Laemmli et al. Nature 227, 680 (1970) as shown in FIG. 4. Samples wereboiled in 2% SDS containing sample buffer with or without 0.7M2-mercaptoethanol, to distinguish reduced (2-mercaptoethanol) fromnonreduced (no 2-mercaptoethanol) proteins, then run on 12.5% acrylamideSDS gels. The gels were stained with coomassie blue to visualize theproteins. The sizes of the proteins were estimated by comparing theirmobilities to the mobilities of protein standards. Mabs 71 and 73separated into light and heavy chains when run under reducingconditions. The heavy chains were approximately 52 KDa. The light chainfor 73 was slightly smaller (28 KDa) than for Mab 71 (28.5 KDa). The Fabfragments also had two chains: 28.3 and 27.3 KDa for Fab 71 and 27.5 and26.5 KDa for Fab 73. When these Fab fragments were run under nonreducing conditions, the sizes of Fabs 71 and 73 were approximately 48and 47 KDa respectively. This indicates that the Fab fragments aremonovalent, the complex has one each of the light and heavy chains. Incontrast the mobilities on nonreducing SDS gels for Mabs 71 and 73indicated that their sizes were approximately 200 KDa. This indicatesthat these Mabs are bivalent, there are two each of the heavy and lightchains.

To see if monovalent Fab 71 fragments would activate the EPO receptor,Mab 71 and the Fab 71 fragment were incubated with UT7-EPO cells andthymidine uptake was measured as described in Example 7. As shown inFIG. 5, both rHuEPO and Mab 71 stimulated thymidine uptake. However themonavalent Fab 71 fragment did not. A control monoclonal antibody raisedagainst an unrelated receptor (Her2/neu) also did not stimulatethymidine uptake. This indicates that the antibodies must be bivalent inorder to activate the receptor.

D. Stimulation of Thymidine Uptake by Mab 71 and Fab 71 in the Presenceof rHuEPO.

The fact that Mab 71 inhibits binding of EPO to EPO receptors suggestedthat the antibody may not activate the EPO receptor in the presence ofEPO. To test this possibility UT7-EPO cells were incubated with 30munits/ml rHuEPO and varying amounts of purified Mab 71, Fab 71 or Mabcontrol (raised against Her2/neu). Thymidine uptake was measured asdescribed above. As shown in FIG. 6 Both Mab 71 and Fab 71 inhibitedthymidine uptake at high doses. However at doses between approximately30 and 3000 μg/ml, Mab 71 stimulated thymidine uptake above levelsstimulated by rHuEPO alone. Fab 71 and control antibodies did not havethis effect. This indicates that Mab 71 and rHuEPO can have an additiveeffect in EPO receptor activation.

EXAMPLE 9 Stimulation of Erythroid Colony Formation by Anti-EPORAntibodies

To see if purified Mab 71 would stimulate formation of erythroid cellsfrom precursors in peripheral blood a BFUe assay was done. To purifyerythroid cell precursors, normal human donors were lymphopheresedaccording to standard protocol. The lymphopheresed cells (250 ml) werewashed with 250 ml Hank's Balanced Salt Solution (HBSS). The cells wereresuspended in HBSS and separated by density centrifugation over agradient(Ficoll-paque) for 30 min at 500×g. The low density cells(LD)were collected from the gradient and washed with 500 ml HBSS andresuspended in PBS supplemented with 0.5% bovine serum albumin and 5 mMEDTA at a concentration of 5×10⁸ cells/ml. The LD cells were thenfurther purified using a CD34 progenitor Cell Isolation Kit (QBend/10)made by Miltenyi Biotech GmbH. In brief cells were tagged with an antiCD34 monoclonal antibody they were then bound to magnetic microspheresaccording to protocol. The tagged cells were next passed throughpre-filled MiniMacs separation columns, the columns were washed and theCD34⁺ cells were then eluted from the column. This process was repeatedonce more to achieve a higher purity of CD34⁺ cells. The in vitro assaywas done on the purified CD34⁺ cells as described by Iscove et. al.(J.Cell. Physiol 8, 309 (1974)) with the following modifications. Theculture medium was obtained from Gibco BRL (Human bone marrow stem cellproliferation kit; Grand Island, N.Y.). To plate out duplicate 1 mlsamples on 35×100 mm tissue culture plates, an excess of 3 ml wasprepared in 17×100 sterile polystyrene tubes. Each tube received 2.5 mlStem Cell Growth medium, 0.1 ml CD34⁺ cells (resuspended at 90,000cells/ml) 0.015 ml Stem Cell Factor (20 μg/ml), and a combination ofsample and Stem Cell Dilution medium equaling 0.385 ml. The tubes werevortexed and allowed to settle to allow bubbles to rise. The contentswere then aliquoted using a 3 ml syringe with a 17×1-½ needle. Theplates were incubated at 37° C. and 10% CO₂ in a humidified tissueculture incubator. Erythroid colonies (orange to red in color) werescored after 21 days. No erythroid colonies were seen in plates lackingEPO or Mab 71. rHuEPO (30 mUnits/plate) gave an excess of 400 coloniesper plate. Mab 71 also produced erythroid colonies. Peak activity wasseen at 2-6 μg/ml. This result indicates that Mab 71 stimulatesformation of erythroid colonies.

The activity of purified Mab 71 was also tested for the ability to formerythroid colonies using serum free growth conditions inmethylcellulose. CD34⁺ cells were isolated as described above andincubated using the serum free growth medium described in co-pending andco-owned U.S. Ser. No. 08/079,719, hereby incorporated by reference,with the following modifications. The assay tubes were set up withoutusing extracellular matrix molecules, hydrocortisone, and the growthfactors EGF, FGF, and PDGF. As described above 3 mL of sample wasprepared to plate out duplicate 1 mL samples on plates. Each tubereceived 0.030ml each of 100× Stock Solutions (2-Mercaptoethanol,nucleosides, cholesterol, Sodium-Pyruvate, Hu-Transferrin, lipids,Hu-Insulin), 0.4 ml deionized BSA (15%), 0.015 ml SCF (20 ug/ml), 0.1 mlCD34+ cells (resuspended at 300,000 cells/ml), 1.080 ml methylcellulose(2.3%), and a combination of sample and IMDM equaling 1.195 ml where thesample did not exceed 150 μl. The plates were then incubated asdescribed above and colonies were scored after 21 days. Erythroidcolonies were observed when grown in the presence of EPO or Mab 71 butnot under conditions lacking these two factors. An example of theerythroid colony types seen is shown in FIG. 7. Colonies incubated with25 munits of rHuEPO looked similar to those grown with 2.1 μg/ml ofpurified Mab 71. Higher doses of rHuEPO gave larger colonies. A doseresponse curve is shown in FIG. 8. Mab 71 had a peak in activity atdoses between 1 and 5 μg/ml. Lower and higher doses resulted in fewererythroid colonies. A control monoclonal antibody raised to Her2/Neu didnot produce any colonies over this dose range. This result indicatesthat the Mab 71 will stimulate the formation of erythroid colonies fromerythroid precursors and that there is not an additional requirement forserum. Thus Mab 71 can stimulate differentiation of erythroid precursorsinto erythroid cells.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1. An antibody or fragment thereof which activates an erythropoietinreceptor.
 2. The antibody of claim 1 wherein the erythropoietin receptoris a mammalian erythropoietin receptor.
 3. The antibody of claim 1wherein the erythropoietin receptor is a human erythropoietin receptor.4. The antibody of claim 1 which is a monoclonal antibody.
 5. Theantibody of claim 1 which is a humanized antibody.
 6. The antibody ofclaim 1 which is a human antibody.
 7. The antibody of claim 1 having adetectable label.
 8. A hybridoma cell line capable of producing themonoclonal antibody of claim
 4. 9. An antibody of fragment thereof whichrecognizes an epitope on an erythropoietin receptor which is recognizedby the monoclonal antibody produced by the hybridoma cell line ATCC[[No. ______]] No. HB 11689 or ATCC [[No. ______]] No. HB
 11690. 10. Theantibody of claim 9 which activates an erythropoietin receptor.
 11. Theantibody of claim 9 wherein the erythropoietin receptor is a humanerythropoietin receptor.
 12. The antibody of claim 9 which is amonoclonal antibody.
 13. The antibody of claim 9 which is a humanizedantibody.
 14. The antibody of claim 9 having a detectable label.
 15. Ahybridoma cell line capable of producing the monoclonal antibody ofclaim
 12. 16. (canceled)
 17. (canceled)
 18. A method of detecting in abiological sample an erythropoietin receptor which is capable of beingactivated, the method comprising the steps of: (a) contacting the samplewith the antibody of claims 1 or 9; (b) detecting the activation of thereceptor by the antibody, thereby determining the presence of anerythropoietin receptor which is capable of being activated.
 19. A kitfor detecting in a biological sample an erythropoietin receptor which iscapable of being activated comprising the antibody of claims 1 or
 9. 20.A method of modulating the endogenous activity of an erythropoietinreceptor in a mammal comprising administering an amount of the antibodyof claims 1 or 9 effective to modulate the activity of the receptor. 21.The method of claim 20 wherein the modulation of the erythropoietinreceptor activity regulates proliferation or differentiation oferythroid progenitor cells.
 22. A method of treating anemia in a patientcomprising administering a therapeutically effective amount of theantibody of claims 1 or
 9. 23. A pharmaceutical composition comprising atherapeutically effective amount of the antibody of claims 1 or 9 in apharmaceutically acceptable adjuvant.
 24. The composition of claim 23wherein the antibody is a monoclonal antibody.
 25. The composition ofclaim 24 wherein the antibody is a humanized antibody.
 26. Thecomposition of claim 24 wherein the antibody is a human antibody.